DNA vaccination is an effective means of protecting experimental animals against infectious pathogens and cancer, and has more recently been used to prevent and reverse autoimmune disease. We have demonstrated that vaccination with DNA encoding TCR Vbeta elements, or with DNA sequences encoding myelin antigens can prevent or reverse experimental autoimmune encephalomyelitis. Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease characterized by T cell-mediated destruction of the insulin-secreting Beta cells in the pancreas. The NOD mouse is an animal model of IDDM in which several autoantigens, including insulin, have been identified. In preliminary studies we have demonstrated that vaccination of NOD mice with DNA encoding an immunodominant peptide of insulin-residues 9-23 of the B chain-protects the animals from developing diabetes. Animals injected intramuscularly with a bacterial plasmid encoding the insulin B peptide show lower disease incidence and delayed onset of disease. Protection is mediated by insulin B (9-23)-specific downregulation of IFN-g. DNA immunization is explored here as a potential therapy in the treatment of autoimmune diabetes. We will rely on our growing experience with DNA vaccination in the prevention and treatment of EAE. These studies will complement the sections on therapy with genes introduced by retroviral vectors in the sections on in vitro gene transfer in Dr. Fathman's project, and studies of epitope spreading using a proteomic approach in Dr. Utz' project. In particular, can we predict efficacious cDNAs using the autoantibody screen with peptide microarrays described in Project 3? These studies will attempt to correlate prevention with epitope selection in EAE as well as the studies described above on IDDM. In Aim 1, Reversal of Hyperglycemia, we will investigate whether diabetes in the NOD mouse can be reversed beginning immunization after the onset of glucosuria using DNA immunization to insulin residues 9-23 of the B chain. In Aims 2 and 3 we will utilize IL-4 or IL-12 p40, or NGF as adjuvants with cDNA vaccination. In Aim 4 we will test DNA vaccination with genes encoding multiple islet cell antigens, including insulin, GAD and hsp65. In Aim 5 we will use protein microarrays to identify potential cDNA vaccination candidates. These studies may lead to new therapies for prevention and treatment of autoimmune disease in man.